Generation of splicing inactive pre-mRNAs

The protein assembly was investigated on splicing-active and splicing-inactive pre-mRNAs.

The PM5 pre-mRNA was used as splicing-active pre-mRNA. It lacks the 3’ splice site and the 3’ exon and has been shown to be well-suited to isolate catalytically active step 1 C complexes (Bessonov et al., 2008). When incubated under splicing conditions with HeLa nuclear extract, PM5 undergoes 5’ splice site cleavage and formation of the intron lariat but no exon ligation takes place.

Two splicing-inactive pre-mRNAs were considered for comparison of the protein assembly on splicing-active (PM5) and splicing-inactive pre-mRNAs: (i) PM5 pre-mRNA lacking the 5’

splice site (5’ss) and, (ii) PM5 pre-mRNA lacking the branch point site (BPS). As the U1 and U2 snRNP bind to the pre-mRNA’s 5’ss and BPS, respectively, during the formation of A complex, the use of pre-mRNAs from which the 5’ss and BPS have been deleted will hamper the spliceosomal assembly. These pre-mRNAs are therefore splicing-inactive and may be expected to prevent spliceosomal complex formation.

The sequence of the PM5 plasmid is shown in Figure 4.27. The nucleotide sequence AGGTATGT or ACTGA (highlighted in red) was deleted to generate 5’ss- or BPS-deleted PM5 pre-mRNAs, respectively. Generation of the pre-mRNAs was performed as outlined in

section 3.2.1.11 using the PM5 plasmid as template. Two PM5 pre-mRNAs, one lacking the 5’ss and one lacking the BPS, respectively, were obtained.

Figure 4.27: Sequence of the PM5 plasmid. MS2 binding sites (MS2), exon 1, branch point site (BPS) and polypytimidine tract (Yn) are shown in bold. Restriction sites of DNA restriction enzymes are shown in blue.

Deleted nucleotide sequences are shown in red.

The kinetics of splicing and spliceosomal complex formation were investigated for PM5 pre-mRNA and 5’ss- and BPS-deleted PM5 pre-pre-mRNAs (Figure 4.28). When PM5 pre-pre-mRNA was used, splicing products (intron lariat and MS2-exon) first appeared after 10 minutes. The amount of pre-mRNA was reduced during incubation. As RNAseH digestion leads to degradation of early spliceosomes, the pre-mRNA disappeared and only the splicing products and an RNAseH digestion product were visible after RNAseH digestion. For PM5 5’ss-deleted pre-mRNA, no splicing products were formed, and the amount of pre-mRNA remained constant. After 180 minutes incubation followed by RNAseH digestion a digestion product was observed. However, using BPS-deleted PM5 pre-mRNA the appearance of splicing products after 30 minutes and 180 minutes followed by RNAseH digestion was indicated (Figure 4.28 A). Spliceosomal complex formation was assayed by native agarose gel electrophoresis, and the complexes formed were visualized by autoradiography. On PM5

pre-mRNA, H/E complexes were rapidly formed, whereas A and B complexes first appeared after 2-4 minutes. C complex formation was first observed after 10 minutes. Surprisingly, A and B complex formation was also observed on PM5 5’ss deleted pre-mRNA, although B complex formation seemed to be delayed about 2 minutes. However, C complex formation was not observed on the 5’ss-deleted PM5 pre-mRNA. Complex formation on BPS-deleted PM5 pre-mRNA suggests also formation of A and B complexes. Strikingly, after 180 minutes incubation followed by RNAseH digestion the presence of C complexes was indicated, whereas this is not clearly seen (Figure 4.28 B).

Figure 4.28: The splicing kinetics and the spliceosomal complex formation using PM5 pre-mRNA and 5’ss- and BPS-deleted PM5 pre-mRNA. (A) Splicing kinetics were followed by denaturing gel electrophoresis.

Radioactively labeled pre-mRNA and splicing products were visualized by autoradiography. With PM5 pre-mRNA, splicing products first appeared after 10 minutes and the amount of pre-mRNA was reduced during incubation.

With 5’ss-deleted PM5 pre-mRNA, no splicing products were observed, whereas the appearance of splicing products after 30 minutes was indicated for BPS-deleted PM5 pre-mRNA. (B) Spliceosomal complex formation was assayed by native agarose gel electrophoresis, and the complexes formed were visualized by autoradiography. On PM5 pre-mRNA, the formation of H/E, A, B and C complexes was observed. Surprisingly, A and B complex formation was also observed on 5’ss-deleted PM5 pre-mRNA. On BPS-deleted PM5 pre-mRNA, A and B complex formation was indicated and after 180 minutes incubation followed by RNAseH digestion C complex formation was suggested.

The splicing kinetics revealed that no pre-mRNA splicing occurred when 5’ss-deleted PM5 pre-mRNA was used. Surprisingly, A and B complex formation was observed, even though the 5’ss, with which the U1 snRNP is known to make contact during A complex formation, was deleted. Nonetheless, C complex formation was not detected, confirming that this pre-mRNA is indeed splicing-inactive. Strikingly, splicing products appeared after 30 and 180 minutes when using BPS-deleted PM5 pre-mRNA. During complex formation the presence of C complex after RNAseH digestion is indicated, although this is not clearly visible. Inspection of the PM5 plasmid sequence revealed that the deleted nucleotide sequence (ACTGA) exists once more in the PM5 plasmid. This duplicate is located 28 nucleotides downstream of the branch point site within the intron of the PM5 pre-mRNA (see Figure 4.27) and might serve as an alternative branch point. This would explain the presence of splicing products that were observed when the splicing kinetics and complex formation were analyzed.

For this reason, an additional pre-mRNA was produced in which the duplicate BPS sequence was also deleted (in the following referred to as “BPS-ACTGA-deleted PM5 pre-mRNA”). The splicing kinetics and the spliceosomal complex formation were then investigated for the BPS- and the BPS-ACTGA-deleted mRNA (Figure 4.29). As described above, when PM5 mRNA was used, splicing products were observed after 10 minutes and the amount of mRNA present decreased during the incubation. The splice assay for BPS-deleted PM5 pre-mRNA again revealed the presence of splicing products after 30 and 180 minutes. The

“double-deleted” pre-mRNA (the BPS-ACTGA-deleted PM5 pre-mRNA) did not show splicing products, confirming that this pre-mRNA mutant is splicing-inactive (Figure 4.29 A).

Spliceosomal complex formation for PM5 pre-mRNA again showed H/E, A, B and C complex formation. As described above, A and B complex formation was also observed for the BPS-deleted PM5 pre-mRNA; moreover C complex formation after 30 and 180 minutes was suggested, although it was not clearly visible. For the BPS-ACTGA-deleted PM5 pre-mRNA no comparable complex formation was achieved. However, native agarose gel electrophoresis indicated the formation of some complexes on this particular pre-mRNA (Figure 4.29 B). The complexes formed differed in their migration behavior in native agarose gel electrophoresis and thus appear to have a different RNA and/or protein composition as compared with the spliceosomal A, B and C complexes. Nonetheless, as the “double-deleted” pre-mRNA (BPS-ACTGA-deleted PM5 pre-mRNA) did not lead to splicing products and did not show C complex formation, it is splicing-inactive. Therefore, in this study the PM5 pre-mRNA and the 5’ss- and the BPS-ACTGA-deleted PM5 pre-mRNAs were used for comparison of the protein assembly on splicing-active and splicing-inactive pre-mRNAs.

Figure 4.29: The splicing kinetics and the spliceosomal complex formation using PM5 pre-mRNA and BPS and BPS-ACTGA deleted PM5 pre-mRNAs. (A) Splicing kinetics were followed by denaturing gel electrophoresis. Pre-mRNA and splicing products were visualized by autoradiography. With PM5 pre-mRNA, splicing products first appeared after 10 minutes and the amount of pre-mRNA was reduced during incubation.

With the deleted pre-mRNA splicing products had appeared after 30 minutes. For the “double-deleted” BPS-ACTGA-deleted PM5 pre-mRNA no splicing products were observed. (B) Spliceosomal complex formation was assayed by native agarose gel electrophoresis and the complexes formed were visualized by autoradiography.

On PM5 mRNA, the formation of H/E, A, B and C complexes was observed. On BPS-deleted PM5 pre-mRNA, A and B complex formation was detected, and after 180 minutes incubation followed by RNAseH digestion some C complex formation was detected. For BPS-ACTGA-deleted PM5 pre-mRNA no comparable complex formation was observed, whereas formation of some complexes is indicated.